Therapeutic regimens for BAFF antagonists

ABSTRACT

Therapeutic regimens for administration of BAFF antagonists for treatment of immunologic and related disorders are described. Regimens involve a short-term BAFF antagonist administration course followed by an extended no-treatment period prior the round of administration.

This application claims priority to U.S. Provisional Application No.60/512,880, filed Oct. 20, 2003, which is hereby incorporated byreference.

FIELD OF THE INVENTION

The present invention generally relates to therapeutic regimens used intreating immunologic and related disorders. More specifically, theinvention relates to methods of treating autoimmune disorders utilizingantagonists of BAFF (B cell activating factor belonging to the TNFfamily).

BACKGROUND OF THE INVENTION

B cells play a central role in acquired immunity. These cells possessthe unique ability to mount a rapid and directed antibody (Ab) responseagainst foreign antigens (Ag), and to act as Ag presenting cells. Tomaintain B cell homeostasis and a self-tolerant state, it is necessaryto generate a continuous pool of B cell precursors that will mature andmigrate to peripheral organs, as well as maintain a process of negativeselection to eliminate autoreactive B cells. Dysregulation in the B celldevelopmental process could lead to a block in B cell development, andthus immune deficiency, or conversely, to an escape and expansion ofself-reactive B cells leading to autoimmunity.

Generation of high affinity, somatically hypermutated autoantibodies isone of the hallmarks of autoimmune conditions. The autoantibodies cancause severe tissue damage (e.g., as in lupus nephritis) or loss ofblood components (e.g., as in immune thrombocytopenia purpura). Theprevailing treatment strategies for autoimmune disorders employ globalimmunosuppressants that have harmful side effects with long-term use.

Recent discovery of the B cell survival and maturation factor BAFF (alsoknown as TALL-1, THANK, BLyS, zTNF4, and TNFSF13B) provided a uniqueopportunity for developing targeted intervention strategies forautoreactive B cell function. Elucidation of the role of BAFF inacquired immunity has been rapid since its first description as a B cellgrowth factor. BAFF (Accession No. AAD25356) is described in, e.g.,Schneider et al (1999) J. Exp. Med., 189:1697-1710; PCT Publication WO99/12964 and U.S. patent application Ser. No. 09/911,777. Studies haveindicated that higher than normal levels of BAFF may contribute to thepathogenesis of autoimmune diseases, such as systemic lupuserythematosus (SLE) and rheumatoid arthritis. For a review, see, e.g.,Mackay et al. (2002) Nature Reviews: Immunology, 2:465-475; Kalled etal. (2003) Expert Opin. Ther. Targets, 7(1):115-23.

BAFF has been implicated in costimulation of B cells (Moore et al.(1999) Science, 285:260-263; Schneider et al. (1999) J. Exp. Med.,189:1747-1756; Mukhopadhyay et al. (1999) J. Biol. Chem.,274:15978-15981); increased B cell proliferation (Moore et al. (1999)Science, 285:260-263); and increased survival of normally deleted Bcells (Khare et al. (2000) Proc. Natl. Acad. Sci., 97:3370-3375; Grosset al. (2000) Nature, 404:995-999; Mackay et al. (1999) J. Exp. Med.,190:1697-1710). Three cognate receptors for BAFF have been identified:(1) B cell maturation antigen (BCMA; Accession No. S43486; Gross et al.(2000) Nature, 404:995-999; PCT Publication WO 01/12812; U.S. patentapplication Ser. No. 10/077,137); transmembrane activator andcyclophilin ligand interactor (TACI; Accession No. AAP57629; Gross etal., supra); and most recently, BAFFR (also called BR3; Accession No.AF373846; Thompson et al. (2001) Science, 293:2108-2111). BAFFR is theonly one of the three receptors that is specific for BAFF (Thompson etal., supra). BCMA and TACI bind not only to BAFF but also to another TNFfamily ligand, APRIL (Yu et al. (2000) Nat. Immunol., 1:252-256; Wu etal. (2000) J. Biol. Chem., 275:35478-35485; Rennert et al. (2000) J.Exp. Med., 192:1677-1684; PCT Publication WO 01/24811; U.S. patentapplication Ser. No. 10/115,192).

While BAFF has been unambiguously identified as a factor required for Bcell survival and maturation, the role of APRIL in B cell homeostasisremains less well understood. APRIL was originally found to stimulategrowth of tumor cells in vitro and in vivo (Hahne et al. (1998) J. Exp.Med., 188:1185-1190). Recent reports demonstrated that APRIL may actalso as a costimulator of primary B and T cells in vitro and stimulateIgM production by peripheral blood B cells in vitro (Yu et al. (2000)Nat. Immunol., 1:252-256; Marsters et al. (2000) Curr. Biol.,10:785-788). In vivo administration of APRIL, like BAFF, results insplenomegaly due to expansion of the B cell population and an increasein the percentage of activated T cells (Yu et al. (2000) Nat. Immunol.,1:252-256), suggesting that APRIL may be involved in lymphoidhomeostasis. Based on data generated using APRIL-transgenic mice, APRILappears to play a role in T cell-independent type 2 (TI-2) humoralresponses (Stein et al. (2002) J. Clin. Invest., 109(12): 1587-1598).Further, data generated using A/WySnJ mice demonstrate that APRIL is notrequired for B cell development and function (Thompson et al., supra).

Soluble forms of BAFF receptors have been made by fusing theirextracellular domains to the Fc portion of immunoglobulin. Treatment ofnormal mice with such a soluble form of TACI or BCMA (TACI-Fc orBCMA-Fc) leads to reduced B cell numbers and a lack of humoral response(Shu et al. (1999) J. Leukoc. Biol., 65:680-683; Yan et al. (2000) Nat.Immunol., 1:37-41; Xia et al. (2000) J. Exp. Med., 192:137-143; Wang etal. (2001) Nat. Immunol., 2:632-637; Yu et al. (2000) Nat. Immunol.,1:252-256). For example, in a mouse model for rheumatoid arthritis, anautoimmune disease that involves both B and T cell components, TACI-Fcsubstantially inhibits inflammation and slows disease progression (Wanget al. (2001) Nat. Immunol., 2(7):632-637). These effects are thought tobe attributed to BAFF sequestration because BAFF-deficient mice have aphenotype similar to that of TACI-Fc- or BCMA-Fc-treated mice (almostcomplete loss of mature B cells and a severely compromised humoralresponse) (Schiemann et al. (2001) Science, 293:2111-2114; Gross et al.(2001) Immunity, 15:289-302). Although a five-week treatment of NZBWF1with TACI-Ig lead to a reduction in SLE symptoms at 12 weeks after thetreatment, no reduction in autoantibodies was observed, indicating thata longer course of treatment may be necessary (Gross et al. (2000)Nature, 404(6781):995-999).

Recently, BAFF-specific agents, including BAFFR-Fc and BAFF antibodies,have been developed for treatment of autoimmune and other disorders(see, e.g., U.S. patent application Ser. Nos. 09/911,777; 10/380,703;10/045,574; and 60/458,707); Kalled et al. (2003) Expert Opin. Ther.Targets, 7(1):115-23). Once the administration of a BAFF-specific agenthas ceased and the agent is eliminated from the bloodstream, B cellreconstitution to pre-treatment levels, including recovery of pathogenicB cells, is expected to occur within about 8 weeks (see, e.g., Porpigliaet al. (2003) Clin. Immunol., 107:90-97; Berardi et al. (1997) Blood,89:3554-3564; Hiramatsu et al. (2003) Blood, 102:873-880). As a result,frequent administration of these drugs, at intervals of less than 8weeks, may be necessary to maintain therapeutic benefits.

Therefore, new therapeutic strategies are still needed for the treatmentof immune and related disorders.

SUMMARY OF THE INVENTION

The present invention results from the realization that effectivetreatment of immunologic disorders can be accomplished by a short-termadministration of a therapeutically effective amount of a BAFFantagonist, thereafter, temporarily discontinuing the administration ofthe antagonist for an extended period prior to the next round of theadministration.

The invention is based, in part, on the surprising finding that ashort-term 4-week administration of BAFFR-Fc to nephritic SNF1 mice withmild-to-moderate SLE results in a long-term clinical benefit (at least 6months). Although B cell recovery does take place followingadministration of BAFFR-Fc, the newly emerging B cells are substantiallynon-pathogenic. The invention is further based, on the discovery anddemonstration that the clinical effect of the short-term BAFFR-Fcadministration in the SNF1 mice is significantly more pronounced andlong-lasting as compared to the clinical effect observed uponadministration of TACI-Fc in the same model.

The present invention provides methods for treating immunologic andrelated disorders in mammals, in particular autoimmune disorders, andincludes (but is not limited to) methods for inhibiting autoantibodyproduction, and/or inhibiting the generation of pathogenic B cellpopulation in a mammal. The invention further provides methods fortreating renal fibrosis and/or improving renal function, such as, e.g.,pressure filtration, selective reabsorption, tubular secretion, andsystemic blood pressure regulation. The invention yet further providesmethods for decreasing cardiac inflammation. Methods of administrationand compositions used in the methods of the inventions are alsoprovided.

In certain embodiments, the method of treating a patient having animmunologic disorder, comprises:

(a) administering to the patient a therapeutically effective amount of aBAFF (B cell activating factor belonging to the TNF family) antagonistat least once or at one or more intervals of less than N weeks;

(b) temporarily discontinuing the administration of step (a) for N weeksor longer; and

(c) repeating steps (a) and (b) at least once;

wherein N is 8, 9, 10, 11, or 12.

In some embodiments, the BAFF antagonist is a soluble BAFF receptor(e.g., BAFFR, BCMA, or TACI), an anti-BAFF antibody, or an anti-BAFFreceptor antibody. In certain embodiments, the BAFF antagonist isBAFF-specific such as, for example, soluble BAFFR.

Additional aspects of the invention will be set forth in part in thefollowing description, and in part will be understood from thedescription or may be learned by practice of the invention.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO:1 is an amino acid sequence of human BAFFR (GenBank™ AccessionNo. AF373846). Special features noted in the Sequence Listing for thissequence: residue 1—none or any amino acid; residue 2—methionine, none,or any amino acid; residue 21—valine (wild type), asparagine, or anotheramino acid; residue 28—lysine (wild type), proline, or another aminoacid; residue 47—none, any amino acid, or alanine.

SEQ ID NO:3 is an amino acid sequence of human BAFFR-Fc fusion protein,which includes a signal sequence (amino acids 1-22) and a human IgG1 Fcportion (amino acids 95-321). Special features noted in the SequenceListing for this sequence: residue 41—valine (wild type), asparagine, oranother amino acid; residue 48—lysine (wild type), proline, or anotheramino acid; residue 67—none, any amino acid, or alanine.

SEQ ID NO:2 is an amino acid sequence of murine BAFFR (GenBank™Accession No. Q96RJ3).

SEQ ID NO:4 is an amino acid sequence of murine BAFFR-Fc fusion protein,which includes a signal sequence (amino acids 1-22) and a murine IgG1 Fcportion (amino acids 88-316).

SEQ ID NO:5 is an amino acid sequence of a GAFF-binding peptide derivedfrom BAFFR.

SEQ ID NO:6 is an amino acid sequence of a minimal deletion in ΔBAFFRdescribed in U.S. Patent Application No. 60/458,707.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1D demonstrate that a short-term BAFFR-Fc treatment inhibitsrenal lymphocyte infiltration in the kidneys of SNF1 mice as assessed byimmunohistochemistry on paraffin-embedded kidney sections usingbiotin-anti-B220 mAb for B cells and anti-CD3 for T cells (50×magnification). FIGS. 1A and 1B show representative kidney sections fromuntreated mice of 9 and 50 weeks of age, respectively. Two groups ofmice were administered either 200 μg of murine BAFFR-Fc or 200 μg ofmIgG1, two times per week for four consecutive weeks starting at 23weeks of age, and sacrificed at the age of 50 or 51 weeks, respectively.FIGS. 1C and 1D show representative kidney sections from BAFFR-Fc- andmIgG1-treated mice, respectively.

FIGS. 2A-2D demonstrate that a short-term BAFFR-Fc treatment inhibitslymphadenopathy in SNF1 mice as assessed by immunohistochemistry onparaffin-embedded mesenteric lymph node (MLN) sections usingbiotin-anti-B220 mAb for B cells and anti-CD3 for T cells (50×magnification). FIGS. 2A and 2B show representative MLN sections fromuntreated mice of 9 and 50 weeks of age, respectively. Two groups ofmice were administered either 200 μg of murine BAFFR-Fc or 200 μg ofmIgG1, two times per week for four consecutive weeks starting at 23weeks of age, and sacrificed at the age of 50 or 51 weeks, respectively.FIGS. 2C and 2D show representative MLN sections from BAFFR-Fc- andmIgG1-treated mice, respectively.

DETAILED DESCRIPTION OF THE INVENTION

In order that the present invention may be more readily understood,certain terms are first defined. Additional definitions are set forththroughout the detailed description.

The term “antibody,” as used herein, refers to an immunoglobulin or apart thereof, and encompasses any polypeptide comprising anantigen-binding site regardless of the source, method of production, andother characteristics. The term includes but is not limited topolyclonal, monoclonal, monospecific, polyspecific, non-specific,humanized, single-chain, chimeric, synthetic, recombinant, hybrid,mutated, and CDR-grafted antibodies. The term “antigen-binding domain”refers to the part of an antibody molecule that comprises the areaspecifically binding to or complementary to a part or all of an antigen.Where an antigen is large, an antibody may only bind to a particularpart of the antigen. The “epitope,” or “antigenic determinant” is aportion of an antigen molecule that is responsible for specificinteractions with the antigen-binding domain of an antibody. Anantigen-binding domain may be provided by one or more antibody variabledomains (e.g., a so-called Fd antibody fragment consisting of a V_(H)domain). An antigen-binding domain comprises an antibody light chainvariable region (V_(L)) and an antibody heavy chain variable region(V_(H)). The terms “anti-BAFF antibody” and “antibody directed againstBAFF” refer to any antibody that specifically binds to at least oneepitope of BAFF.

The term “BAFF” refers to B cell-activating factor, characterized byexpression in immune cells and its role as a B cell survival factor. Asummary of BAFF's characteristics is provided in Mackay et al. (2002)Nature Reviews: Immunology 2:465-475 and in Gavin et al. (2003) J. Biol.Chem., 278(40):38220-8.

As used herein, “BAFF antagonist” generally refers to any compound thatdirectly downregulates the biological activity of BAFF. A molecule“directly downregulates” the biological activity of BAFF by interactingwith a BAFF; BAFF gene, a BAFF transcript, or a BAFF receptor. A BAFFantagonist may, for example, bind to and neutralize the activity ofBAFF; decrease BAFF expression levels; affect stability of BAFF; affectproteolytic cleavage of the membrane-bound form of BAFF into the solubleform; interfere with the binding of BAFF to one or more receptors; or itmay interfere with intracellular signaling of a BAFF receptor. BAFFantagonists may be proteinaceous (e.g., antibodies, receptor fusionproteins, peptides) or non-proteinaceous molecules (e.g., small organicmolecules (≦500 Da)). Methods for assessing neutralizing biologicalactivity of BAFF antagonists are known in the art.

The term “BAFFR,” unless otherwise stated, refers to a protein thatcomprises at least a portion of wild-type or mutant receptor for BAFF,other than BCMA or TACI, that is capable of binding to BAFF. It has beendetermined that the BAFF-binding domain of human BAFFR contains aminoacids 27 to 32 of SEQ ID NO:1. BAFFR is further defined in PCTPublication WO 02/24909 and U.S. patent application Ser. Nos. 10/380,703and 60/458,707, and specifically includes, but is not limited to, humanBAFFR (SEQ ID NO:1; Accession No. AAD25356; amino acid 47 of SEQ ID NO:1is not present in some isoforms) and murine BAFFR (SEQ ID NO:3;Accession No. Q96RJ3). “BAFFR” also refers to naturally occurringvariants, e.g., the splice variant containing an alanine at amino acid47 of SEQ ID NO:1 corresponding to amino acid 67 of SEQ ID NO:2, as wellas functional mutated forms of BAFFR, e.g., human BAFFR having mutationsat least at amino acids 21 and 28 of SEQ ID NO:1 (e.g., V21 N and L28P).The terms “BAFFR-Fc” and “BAFFR-Ig” refer to a fusion protein comprisingBAFFR and antibody constant region sequences, such as, for example, anFc portion.

The term “BAFF-specific antagonist” refers to a compound that: (1) hasthe ability to counteract the effect(s) of BAFF in vivo or in vitro,e.g., by competitive blockage of one or more BAFF receptors, and (2)under physiologic conditions preferentially forms a relatively stablecomplex with BAFF but not with other ligands of the TNF family, such as,e.g., APRIL. Typically, the binding is considered specific when theaffinity constant Ka for BAFF is higher than 106 M-1, preferably higherthan 108 M-1, while the affinity for another TNF family ligand is lowerthan 106 M-1, preferably lower than 105 M-1. A skilled artisanrecognizes that under certain conditions a low affinity but high aviditybinding may also be specific even though Ka of the interaction may berelatively low. In some embodiments, affinity constant Ka of aBAFF-specific antagonist for at least one isoform of BAFF is preferablygreater than 106 M-1, 107M-1, 108 M-1, 109 M-1, 1010 M-1, 1011 M-1, or1012 M-1.

The phrase “substantially identical” means that a relevant amino acidsequence is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or100% identical to a given sequence. By way of example, such sequencesmay be variants derived from various species, or they may be derivedfrom the given sequence by truncation, deletion, amino acid substitutionor addition. Percent identity between two amino acid sequences may bedetermined by standard alignment algorithms such as, for example, BasicLocal Alignment Tool (BLAST) described in Altschul et al. (1990) J. Mol.Biol., 215:403-410, the algorithm of Needleman et al. (1970) J. Mol.Biol., 48:444-453, or the algorithm of Meyers et al. (1988) Comput.Appl. Biosci., 4:11-17. Such algorithms are incorporated into theBLASTN, BLASTP, and “BLAST 2 Sequences” programs (seewww.ncbi.nlm.nih.gov/BLAST). When utilizing such programs, the defaultparameters can be used. For example, for nucleotide sequences thefollowing settings can be used for “BLAST 2 Sequences”: program BLASTN,reward for match 2, penalty for mismatch −2, open gap and extension gappenalties 5 and 2 respectively, gap x_dropoff 50, expect 10, word size11, filter ON. For amino acid sequences the following settings can beused for “BLAST 2 Sequences”: program BLASTP, matrix BLOSUM62, open gapand extension gap penalties 11 and 1 respectively, gap x_dropoff 50,expect 10, word size 3, filter ON.

The term “immunologic disorder” refers to disorders and conditions inwhich an immune response is aberrant. The aberrant response can be dueto (a) abnormal proliferation, maturation, survival, differentiation, orfunction of immune cells such as, for example, T or B cells. Example ofimmunologic disorders include, but are not limited to, rheumatoidarthritis (RA), asthma, psoriasis, multiple sclerosis (MS), inflammatorybowel disease (IBD), Crohn's disease, systemic lupus erythematosis(SLE), type I diabetes, Wegener's granulomatosis, transplant rejection,graft-versus-host disease (GVHD), hyperproliferative immune disorders,autoimmune diseases, B cell cancers, immunomodulation, antibody-mediatedpathologies (e.g., ITCP, myasthenia gravis, and the like), and Sjogren'ssyndrome. The disorders that can be treated using the compositions andmethods of the present invention include but are not limited todisorders described in PCT Publication WO 02/24909 and U.S. patentapplication Ser. Nos. 09/911,777; 10/380,703; 10/045,574; and60/458,707.

The term “therapeutic” and cognates thereof, as used herein, refer toamelioration of clinical manifestations of a disorder, or production ofa desired biological outcome. Examples of desirable outcomes include:reduced B cell hyperplasia; reduced autoantibody titer; reducedpathogenic B cell population; reduced cardiac inflammation; improvingrenal function (e.g., pressure filtration, selective reabsorption,tubular secretion, and systemic blood pressure regulation), slowedprogression renal fibrosis, reduced renal lymphocyte infiltration;reduced lymphadenopathy, etc. Outcomes can be determined using methodsknown in the art and/or as described herein.

The term “pathogenic B cell” refers to a B cell that producesautoantibody which contributes to disease pathology.

The invention is based, in part, on the surprising finding that ashort-term 4-week administration of BAFFR-Fc to nephritic SNF1 mice withmild-to-moderate systemic lupus erythematosus (SLE) results in along-term clinical benefit (at least 6 months). Given a shortcirculatory half-life of BAFFR-Fc in SNF1 mice (3.7 days), once BAFFR-Fcadministration has ceased and BAFFR-Fc is eliminated from thebloodstream, B cell reconstitution is expected to occur within about 8weeks. In experiments leading to the present invention, B cell recoverydid take place following administration of BAFFR-Fc. However, the newlyemerging B cells were found to be substantially non-pathogenic. Theinvention is further based, on the discovery and demonstration that theclinical effect of the short-term BAFFR-Fc administration in the SNF1mice is significantly more pronounced and long-lasting as compared toTACI-Fc in the same model.

Methods of the inventions, compositions used therein, and other aspectsof the invention are described in detail below.

Methods

The invention provides therapeutic regimens that involve a short-termBAFF antagonist administration course followed by an extendedno-treatment period prior to the next round of administration.

In one aspect, the invention provides a therapeutic regimen for treatinga patient having an immunologic or a related disorder. The regimencomprises:

(a) administering to the patient a therapeutically effective amount of aBAFF (B cell activating factor belonging to the TNF family) antagonistat least once or at one or more intervals of less than N weeks;

(b) temporarily discontinuing the administration of step (a) for N weeksor longer; and

(c) repeating steps (a) and (b) at least once;

wherein N is 8, 9, 10, 11, or 12.

In further embodiments, the administration of step (a) comprises aninterval of 1, 2, 3, 4, 5, 6, or 7 weeks. In step (a), the BAFFantagonist may, for example, be administered 2, 3, 4, 5, 6, or 7 times aweek, weekly, biweekly, every 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks.In the administration period of step (a), the intervals betweensequential administrations as well as dosages may vary. For example, inthe administration period of step (a), the BAFF antagonist may beadministered twice in the first week, withdrawn for three weeks,administered once, withdrawn for next 5 weeks, then administered dailyfor a week. In various embodiments, the administration is temporarilydiscontinued, as in step (b), for 8, 9, 10, 11, 12, 18, 24, 30, 36, 42,48 weeks or a longer period which may be limited to 50, 80, 110, 140,180, 210, 260, or 300 weeks.

In one aspect, the invention provides methods for treating immunologicand related disorders in mammals, in particular autoimmune disorders,and includes (but is not limited to) methods for reducing autoantibodytiter (e.g., anti-nuclear antibodies such as, for example, anti-ssDNA,anti-Sm, anti-RNP, and anti-Ro).

In another aspect, the invention further provides methods for treatingrenal fibrosis and/or improving renal function, such as, e.g., pressurefiltration, selective reabsorption, tubular secretion, and systemicblood pressure regulation. In yet another aspect, the invention yetfurther provides methods for decreasing cardiac inflammation.

The invention further provides a method of reducing a pathogenic B cellpopulation in a patient having an immune disorder. In certainembodiments, the pathogenic population of B cells is composed of B cellsthat are IgM-IgD+. B cell populations can be determined using, forexample, antibodies to commonly used B cell markers such as, e.g., theB220 isoform of CD45 (Kincade et al. (1981) J. Immunol., 127:2262-2268), or CD19 (see e.g., Krop et al. (1996) Eur. J. Immunol.,26:238-242) and respective isotype anti-immunoglobulin antibodies usingmethods known in the art and/or as described in the Examples.

The compositions of the invention may be administered in any manner thatis medically acceptable. “Administration” is not limited to anyparticular delivery system and may include, without limitation,parenteral (including subcutaneous, intravenous, intramedullary,intraarticular, intramuscular, or intraperitoneal injection) rectal,topical, transdermal, or oral (for example, in capsules, suspensions, ortablets). Administration to an individual may occur in a single dose orin repeat administrations, and in any of a variety of physiologicallyacceptable salt forms, and/or with an acceptable pharmaceutical carrierand/or additive as part of a pharmaceutical composition (describedearlier). Physiologically acceptable salt forms and standardpharmaceutical formulation techniques and excipients are well known topersons skilled in the art (see, e.g., Physicians' Desk Reference (PDR)2003, 57th ed., Medical Economics Company, 2002; and Remington: TheScience and Practice of Pharmacy, eds. Gennado et al., 20th ed,Lippincott, Williams & Wilkins, 2000).

Administration of an antagonist to an individual may also beaccomplished by means of gene therapy, wherein a nucleic acid sequenceencoding the antagonist is administered to the patient in vivo or tocells in vitro, which are then introduced into a patient, and theantagonist (e.g., antisense RNA, soluble BAFF receptor) is produced byexpression of the product encoded by the nucleic acid sequence.

In certain embodiments, the patient to be treated using the methods ofthe invention has mild, moderate, or severe disease. In variousembodiments, patients are those having an immunologic disorder and/orare at increased risk for developing renal insufficiency. The treatmentmay be particularly useful in patients with proteinuria of over 1, 1.2,1.5, 1.7, 2.0 g per a 24-hour period, and/or serum creatinine levels of1.0, 1.5, 2.0, 2.5 mg/dl or higher. Other indications include creatinineclearance levels of lower than 97, 90, 80, 70, 60 (men) and 88, 80, 70,60 (women) ml/min, blood urea of 20, 25, 30 mg/dl or higher, and/orthose indicated by renal imaging (e.g., MRI, ultrasound), or renalbiopsy. Yet other indications include above normal titer of antinuclearantibodies in the serum (e.g., anti-ssDNA, anti-Sm, anti-RNP, andanti-Ro) or above normal peripheral blood B cell (e.g., CD19+) counts ofover 700, 1000, 1250, 1500, 2000 cells/μl. Tests for assessing theseparameters can be preformed using methods known in the art, as describedin the Examples, or in Jacobs et al. (eds.) (2002) Laboratory testhandbook: Concise with disease index (2nd ed.) Husson, Ohio, Lexi-Comp.

The methods of the invention may be also used for treating patienthaving immunologic disorders where there is abnormal proliferation,maturation, survival, differentiation, or function of immune cells, suchas, for example, B or T cells. The methods of the invention can beuseful for treating pre-B or B cell leukemias, such as plasma cellleukemia, chronic or acute lymphocytic leukemia, myelomas (e.g.,multiple myeloma, plasma cell myeloma, endothelial myeloma, and giantcell myeloma), and lymphoma (e.g., non-Hodgkin's lymphoma), Burkitt'slymphoma, non-Burkitt's lymphoma, follicular lymphoma, acutelymphoblastic leukemia, large cell lymphoma (e.g., immunoblasticlymphoma), marginal zone lymphoma, mantle cell lymphoma, smalllymphocytic lymphoma, other B cell lymphomas, glomerulonephritisassociated with diseases such as membranous nephropathy, IgA nephropathy(Berger's disease), IgM nephropathy, Goodpasture's disease,post-infectious glomerulonephritis, mesangioproliferative disease,chronic lymphoid leukemia, and minimal-change nephritic syndrome,secondary glomerulonephritis, vasculitis, pyelonephritis,nephrocalcinosis, nephrolithiasis, chronic or acute interstitialnephritis, hypertensive or large vessel diseases (including renal arterystenosis or occlusion and cholesterol emboli or renal emboli), renal orurological neoplasms, multiple myelomas, lymphomas, light chainneuropathy, amyloidosis, asthma and other chronic airway diseases suchas bronchitis and emphysema, insulin dependent diabetes mellitus, andCrohn's disease. The methods of the invention can also be used forinhibiting or neutralizing an effector T cell response and for treatingconditions requiring immunosuppression, such as graft versus hostdisease and graft rejection, chronic inflammatory diseases, inparticular, to lessen joint pain, swelling, anemia, and septic shock.

In the methods of the invention, the antagonist(s) is (are) administeredas the sole active compound(s) or in combination with other compound(s)or composition(s). Unless otherwise indicated, the dose of antagonistsadministered is approximately 1 μg/kg and 250 mg/kg, depending on theseverity of the symptoms and the progression of the disease. Mostcommonly, antibodies or immunoglobulin fusion proteins are administeredin an outpatient setting by weekly administration of about 0.1-10 mg/kgdoses by slow intravenous (IV) infusion. The appropriate therapeuticallyeffective dose of an antagonist is selected by a treating clinician andwould range approximately from 1 μg/kg to 100 mg/kg, 1 μg/kg to 50mg/kg, 1 μg/kg to 20 mg/kg, 1 μg/kg to 10 mg/kg, 1 μg/kg to 1 mg/kg, 10μg/kg to 1 mg/kg, 10 μg/kg to 100 μg/kg, 100 μg to 1 mg/kg, or 500 μg/kgto 5 mg/kg. Alternatively, specific dosages indicated in the Examples orfor similar drugs in the Physicians' Desk Reference (PDR) 2003, 57thed., Medical Economics Company, 2002, may be used.

Compositions

BAFF antagonists used in the methods of the present invention, include(but are not limited to) antibodies directed against BAFF, antibodiesdirected against one or more isoforms of at least one BAFF receptor, andsoluble forms of BAFF receptors.

BAFF receptors include BAFFR, BCMA, and TACI. In some embodiments, theBAFF antagonist is BAFF-specific (e.g., BAFFR), while in certain otherembodiments the BAFF antagonist my also bind TNF family ligands otherthan BAFF (e.g., BCMA and TACI which also bind to APRIL). In someembodiments, the BAFF antagonist is an antibody that blocks BAFF bindingto its receptor. Antibodies directed to BAFF and to BAFF receptors havebeen previously described. Producing such antibodies is well within theskill a person skilled in the art (see, e.g., Antibody Engineering, ed.Borrebaeck, 2nd ed., Oxford University Press, 1995). Examples ofantibodies for use in the methods of the invention include thosedescribed in PCT Publication WO 99/12964 and U.S. patent applicationSer. No. 09/911,777) and the anti-BAFF antibody LymphoStat-B™ (HumanGenome Sciences, Rockville, Md.). In further embodiments, the antibodyof the invention may specifically bind, besides BAFF or BAFFR, anotherprotein that is substantially identical to BAFF or BAFFR, respectively.In yet further embodiments, antibodies are directed against BCMA and/orTACI. Also contemplated for use in humans are humanized forms andderivatives of nonhuman antibodies derived from any vertebrate species.

Other examples of antibodies for use in the methods of the inventioninclude mouse monoclonal antibodies produced by hybridoma clone #2.1,deposited as ATCC No. PTA-3689, or by clone #9.1, deposited as ATCC No.PTA-3688, or humanized chimeric, or fully human derivatives thereof.

Soluble forms of BAFF receptor fusion proteins may comprise aBAFF-binding domain of BAFFR, BCMA, and/or TACI. A BAFF-binding domainis located within the extracellular domains (ECD), i.e., the portion ofthe protein normally present on the exterior of a cell expressing theprotein. In some embodiments, the soluble BAFFR is a disulfide-linkedpeptide having the sequence CHWDLLRHWVC (SEQ ID NO:5) (Kayagaki et al.(2002) Immunity, 10:515-524), or a polypeptide comprising this sequence.In yet other embodiments, the soluble BAFFR is a polypeptide comprisingamino acids 27 to 32 or 18 to 43 of SEQ ID NO:1.

In certain embodiments, a soluble form a BAFF receptor comprises aBAFF-binding domain of a BAFF receptor fused to a constant region of animmunoglobulin, i.e., as in BAFFR-Fc. In some embodiments, BAFFR-Igcomprises residues 3 to 73 of SEQ ID N0:1 fused to the Fc portion ofIgG. In illustrative embodiments, BAFFR-Fc comprises SEQ ID N0:2 (human)or SEQ ID N0:4 (murine). In some embodiments, BAFFR is a human BAFFRhaving a C-terminal deletion starting from amino acid 51 of SEQ ID N0:1,which results in an altered O-linked glycosylation pattern (e.g., ΔBAFFRdescribed in United Stated Patent Application No. 60/458,707). In someembodiments, soluble BAFFR comprises ΔBAFFR which lacks at least thesequence of SEQ ID NO:6 (corresponding to amino acids 51-57 of SEQ IDNO:1).

The BAFF-binding domain of BAFFR comprises amino acids (aa) 8 to aa 50,aa 13 to aa 50, or aa 13 to aa 43, or aa 18 to aa 43 of SEQ ID NO:1. Incertain embodiments, the BAFF-binding domain is identical orsubstantially identical to aa 2 to aa 63 of SEQ ID NO:1 or to aa 2 to aa62 of SEQ ID NO:3, including sequences that have been truncated ormutated so long as such sequences retain the ability to bind BAFF. Inillustrative embodiments, BAFFR is a murine sequence as set out from aa2 to aa 66 of SEQ ID NO:3. In other embodiments, BAFFR comprises atleast 20, 25, 30, 35, 40, 45, or 50 contiguous amino acids of SEQ IDNO:1.

In certain embodiments, the constant region of an immunoglobulincomprises any one of CH1, CH2, or CH3 constant regions, or the entire Fcportion (that includes CH2, or CH3), with or without a hinge region. Inmore embodiments, the second amino acid sequence is derived from the Fcportion of an IgG. In related embodiments, the Fc portion is derivedfrom IgG1, IgG4, or another IgG isotype. In illustrative embodiments,the contract region of an immunoglobulin comprises a sequence from aa 95to aa 321 of SEQ ID NO:3, or aa 88 to aa 316 of SEQ ID NO:4. The secondamino acid sequence may comprise the Fc portion of human IgG1, whereinthe Fc is modified to minimize the effector function. Such modificationsinclude changing specific amino acid residues that might alter aneffector function such as Fc receptor binding (Lund et al. (1991) J.Immun., 147:2657-2662 and Morgan et al. (1995) Immunology, 86:319-324),or changing the species from which the constant region is derived.Immunoglobulins may have mutations in the CH2 region of the heavy chainthat reduce effector function, i.e., Fc receptor binding and complementactivation. For example, immunoglobulins may have mutations such asthose described in U.S. Pat. Nos. 5,624,821 and 5,648,260. In the IgG1or IgG2 heavy chain, for example, such mutations may be made at aminoacid residues corresponding to amino acids 234 and 237 in thefull-length sequence of IgG1 or IgG2. Antibodies andimmunoglobulin-receptor fusion proteins may also have mutations thatstabilize the disulfide bond between the two heavy chains of animmunoglobulin, such as mutations in the hinge region of IgG4, asdisclosed in Angal et al. (1993) Mol. Immunol., 30:105-108.

In certain embodiments, a BAFF-binding domain is fused at the C-terminusor the N-terminus, with or without a linker sequence, to the C-terminusor the N-terminus of the constant region of an immunoglobulin. The exactlength and sequence of the linker and its orientation relative to thelinked sequences may vary. The linker may, for example, comprise one ormore Gly-Ser. The linker may be 2, 10, 20, 30, or more amino acid longand is selected based on properties desired such as solubility, lengthand steric separation, immogenicity, etc. It will be understood by oneof ordinary skill in the art that certain amino acids in a sequence ofany protein may be substituted for other amino acids without adverselyaffecting the activity of the protein. It is thus contemplated thatvarious changes may be made in the amino acid sequences of BAFF receptorof the invention, or DNA sequences encoding therefore, as provided,without appreciable loss of their biological activity or utility.

The use of derivatives and analogs of BAFF receptors are also within thescope of the present invention. The derivatives or analogs should befunctionally active, i.e., capable of exhibiting one or more activitiesassociated with a ligand-binding domain of the wild-type BAFFR.Derivatives or analogs that retain this binding ability, or inhibitbiological activity of BAFF can be produced and tested by proceduresknown in the art and/or as described in the Examples. Methods ofproducing such derivatives and analogs include recombinant and syntheticmethods (see, e.g., Maniatis (1990) Molecular Cloning; A LaboratoryManual, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y., and Bodansky et al. (1995) The Practice of Peptide Synthesis, 2nded., Spring Verlag, Berlin, Germany).

The following examples provide illustrative embodiments of theinvention. While the representative procedures are performed in mice, askilled artisan recognizes that they can be successfully performedwithin parameters clinically feasible in human subjects. The skilledartisan will recognize the numerous modifications and variations thatmay be performed without altering the spirit or scope of the presentinvention. Such modifications and variations are encompassed within thescope of the invention. The examples do not in any way limit theinvention.

EXAMPLES

The following describes materials and methods used in the representativeprocedures and experiments described in the subsequent Examples.

Mice

Female (SWR xNZB)F1 (SNF1) mice were bred and maintained as previouslydescribed in, e.g., Gavalchin et al. (1987) J. Immunol., 138:138-148;Datta et al. 1989 Clin. Immunol. Immunopathol., 51:141-56.

Protein Reagents

An expression vector containing murine BAFFR-mulgG1 (BAFFR-Fc) wasconstructed by ligating a Not1+Aat2 fragment containing a signalsequence from a murine Ig-k gene, an Aat2+Sal1 fragment containing thesequence encoding aa 2 to aa 66 of murine BAFFR (Accession No. AF373847)and a Sal+Not1 fragment containing the sequence encoding aa 226 to aa478 of murine IgG1 (Kabat system of numbering) into the Not1 site of amodified version (without the EBNA-1 gene) of pCEP4 (Invitrogen,Carlsbad, Calif.), called CH269. The vector was transfected into 293cells containing the EBNA-1 gene using Lipofectamine™ according to themanufacturer's protocol (Invitrogen). The transfection was maintained inDulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetalcalf serum (FCS), 2 mM glutamine, 100 U/ml penicillin, 100 μg/mlstreptomycin and 250 μg/ml G418. Medium was harvested every 4-7 days forseveral weeks. The conditioned medium was diluted with three volumes ofhigh salt and high pH buffer (1.5 M glycine pH 8.9, 3 M NaCl) toincrease the affinity of mBAFFR-mIgG for protein A. The BAFFR-Fc proteinwas purified by affinity chromatography (protein A) followed by sizeexclusion chromatography. Mouse IgG1 (mIgG1), produced by the MOPC21hybridoma was purified in a similar fashion and was used as a negativecontrol.

Treatment Protocol

Twenty 23 week-old female SNF1 mice with established nephritis(proteinuria of 30-100 mg/dL) were divided into two cohorts of tenmice/group. One cohort received 200 μg of mouse BAFFR-Fc and the otherreceived 200 μg of mIgG1, 2 times per week for 4 consecutive weeks, andmonitored thereafter for progression of disease. Mice that becamemoribund were euthanized, and tissues and blood samples were collectedfor analysis prior to sacrifice. Blood was also collected via theretro-orbital sinus at regular intervals for obtaining serum forautoantibody determination. At termination of the study, kidneys,hearts, and mesenteric lymph nodes (MLN) were placed in 10% neutralbuffered saline and embedded into paraffin blocks for histologicanalysis. The spleens were collected and used for flow cytometry.

Flow Cytometry

Single cell suspensions were made of splenocytes by mechanicaldisruption. Erythrocytes were lysed in an ammonium chloride solution,and cells were then washed 3 times, filtered through a 100-micronfilter, and counted using a hemocytometer. Splenocytes were stained witha fluorochrome-conjugated anti-B220 mAb to detect B lymphocytes asdescribed in Thompson et al. (2001) Science, 293:2108-2111.

Histology

Paraffin-embedded mesenteric lymph nodes (MLN) and kidney sections wereassessed lymphocyte infiltration using rat anti-B220 mAb for B cells andrat anti-CD3 mAb for T cells, followed by biotinylated rabbit anti-ratand avidin-biotin-HRP solution, and visualized using the substrate DAB(Vector Laboratories, Burlingame, Calif.). Kidney fibrosis was assessedby staining kidneys with Mason's trichrome stain and biotin-labeledanti-α smooth muscle actin followed by incubation with streptavidin-HRPand visualized with DAB substrate. Slides were counterstained withMayer's hematoxylin. Heart inflammation was assessed by examination ofH&E-stained heart tissues.

Assessment of Renal and Cardiac Disease

The urine of each mouse was monitored weekly with Albustix™ (BayerCorp., Research Triangle Park, N.C.) to measure proteinuria. H&E-stainedkidney sections were used for histologic examination and scored for:glomerular hypercellularity, enlargement, crescents, mesangialthickening, loops, fibrinoid necrosis and hyaline; interstitialsclerosis, infiltrate and vasculitis; and tubule vasculitis, atrophy andcasts. Heart tissues were examined for vascular and cardiac inflammationand cardiac vacuolation. The grades of 0-4+ were given based on percentinvolvement of the structure being examined, wherein 0=none and 4+ is100% involvement.

ELISA

The ELISA assay used to detect serum anti-ssDNA antibody was performedas described in Kalled et al. (1998) J. Immunol., 160:2158-2165.

Example 1 Short-Term BAFFR-Fc Administration Increases survival

As shown in Table 1, a short-course of BAFFR-Fc treatment markedlyenhanced survival when compared to mIgG control-treated mice. At age 49weeks, all BAFFR-Fc-treated mice were alive, while only 30% of controlmice remained alive.

TABLE 1 Percent survival Age Treatment (weeks) BAFFR-Fc mlgG 23 100 10029 100 100 30 100 90 31 100 90 32 100 80 33 100 70 34 100 30 49 100 30

Example 2 Short-Term BAFFR-Fc Administration Reduces Progression ofNephritis

Renal function was assessed by proteinuria determination at regularintervals. As shown in Table 2, progression of disease to severenephritis (proteinuria≧300 mg/dL) was significantly reduced inBAFFR-Fc-treated mice when compared to mIgG-treated control mice. By age49 weeks, all control mice developed severe nephritis, while noBAFFR-Fc-treated animal had severe nephritis at that age. In a separateexperiment, SNF1 mice were treated with human IgG or with human TACI-Fc(a receptor-Fc fusion protein that binds to both BAFF and APRIL) at 200μg twice per week for four weeks. Although TACI-Fc administrationsomewhat reduced the incidence of severe nephritis initially, eventuallyall TACI-Fc-treated mice developed severe nephritis similarly tocontrols (Table 2).

TABLE 2 Percent incidence of severe nephritis Treatment Age Human Human(wks) BAFFR-Fc mlgG TACI-Fc IgG 23 0 0 0 0 27 0 30 34 60 30 0 70 60 8036 0 90 100 100 49 0 100 100 100

Renal disease was further assessed by an histologic approach. H&Estained kidney sections were scored for renal disease by combining thescores of the individual disease parameters described in Materials andMethods. As seen in Table 3, BAFFR-Fc-treated mice had significantlyreduced cumulative disease scores when compared to mIgG-treated oruntreated controls, and the scores of BAFFR-Fc-treated mice were closerto that of young SNF1 mice, prior to disease onset.

TABLE 3 Cumulative kidney disease score Age (mean ± SD) (weeks) BAFFR-Fcuntreated mlgG 9    1 ± 0 (n = 3) ¹ 49-51 6.3 ± 2.5 (n = 10) 24.3 ± 8.6(n = 4) 23.9 ± 8.6 (n = 7) ¹ n indicates the number of mice used inanalysis.

In SLE-nephritis, renal fibrosis often occurs and may be mediated bysecretion of extracellular matrix (ECM) proteins, α-smooth muscle actin(ASMA) and collagen, by fibroblasts and mesangial cells. ECM productionwas significantly (p<0.05, Student's t test) diminished in mice treatedwith BAFFR-Fc as compared to untreated and mIgG-treated SNF1 mice, asobserved by reduced ASMA (Table 4) and collagen (Table 5) deposition inthe kidneys. Young, 9 week-old SNF1 mice were used as pre-diseasecontrols.

TABLE 4 Percent glomeruli exhibiting collagen deposition¹ Age (mean ±SD) (weeks) BAFFR-Fc untreated mlgG 9 0 (n = 3) 49-51 3.3 ± 8.8 (n = 10)48.2 ± 36.2 (n = 4) 68.8 ± 33.2 (n = 7) ¹70-111 glomeruli were examinedfor each mouse.

TABLE 5 Percent glomeruli exhibiting ASMA production¹ Age (mean ± SD)(weeks) BAFFR-Fc untreated mlgG 9 0 (n = 3) 49-51 8.5 ± 10.9 (n = 10)57.5 ± 29.5 (n = 4) 68.5 ± 32.1 (n = 7) ¹70-111 glomeruli were examinedfor each mouse.

Healthy kidneys do not harbor lymphoid clusters. However, accompanyingdisease in this model of SLE-nephritis, as well as in human disease, isinfiltration of leukocytes into the kidney, including B and T cells,forming follicular-like structures. Therefore, kidney sections wereexamined for the presence of T and B cell infiltrates. In untreated(FIG. 1B) and mIgG-treated (FIG. 1D) mice, large areas of T and B cellinfiltrates that mimic white pulp areas of secondary lymphoid tissueswere observed throughout the kidneys. In contrast, in kidneys of micethat received short-term BAFFR-Fc treatment (FIG. 1C), infiltration of Bcells, as well as T cells, was markedly reduced (FIG. 1). Few or no B orT cells were observed in healthy, 9 week-old (pre-disease) SNF1 mice(FIG. 1A).

Example 3 Short-Term BAFFR-Fc Administration Inhibits CardiacInflammation

H&E-stained paraffin sections of hearts were examined for vascular andcardiac inflammation, and cardiac vacuolation. As seen in Table 6, SNF1mice that received BAFFR-Fc had an overall markedly reduced cardiacscore when compared to mIgG and untreated control mice.

TABLE 6 Cardiac score Age (mean ± SD) (weeks) BAFFR-Fc untreated mlgG 90 (n = 3) ¹ 49-51 1.2 ± 0.4 (n = 10) 2.5 ± 1.1 (n = 4) 4.7 ± 1.9 (n = 3)¹ n indicates the number of mice used in the analysis.

Example 4 Short-Term BAFFR-Fc Administration Prevents B Cell Hyperplasia

Based on a study of BAFF blockade in normal BALB/c mice with BAFFR-Fc,100 μg once a week for 4 consecutive weeks, peripheral B cell numbersare known to be fully restored within about 9 weeks after the last doseof BAFFR-Fc (data not shown). The number of splenic B cells in the SNF1mice was determined 26-28 weeks after the last dose of BAFFR-Fc or mIgG.As seen in Table 7, SNF1 mice that received a short course of BAFFR-Fctreatment had a significantly reduced (p<0.05, Student's t test) splenicB cell number than untreated or mIgG-treated control mice. With time,control mice developed severe splenic B cell hyperplasia. In contrast,BAFFR-Fc-treated mice exhibited a long-term benefit wherein at the endof the study, splenic B cell counts were between that of pre-disease, 9week-old SNF1 mice, and 20 week-old SNF1 mice that have begun toexperience B cell expansion.

TABLE 7 Age Number of splenic B cells (×10⁶) (weeks) BAFFR-Fc untreatedmlgG 9 34.3 ± 9 (n = 3) 20 123 (n = 1) 49-51 77.4 ± 41.7 (n = 10) 197.4± 62 (n = 4) 141 ± 70.6 (n = 3)

In addition, immunohistochemical analysis of the B and T cellorganization in MLN demonstrated that untreated (FIG. 2B) andmIgG-treated (FIG. 2D) mice had extensive B cell expansion, resulting inlymphadenopathy. In contrast, BAFFR-Fc-treated mice (FIG. 2C), did notexhibit B cell hyperplasia and looked similar to young SNF1 mice priorto disease onset (FIG. 2A).

Example 5 Short-Term BAFFR-Fc Administration Inhibits IncreasingProduction of Autoantibody

Sera obtained from SNF1 mice at regular intervals were analyzed forcirculating anti-ssDNA antibody using an ELISA format. When examiningthe change of anti-ssDNA levels with time, from the start of treatmentto age 37 weeks, there is a significant change (increase) in anti-ssDNAin the mIgG-treated mice (p<0.05 using the Student-Newman-Keuls method),however, there is no significant change in autoantibody levels in micethat received BAFFR-Fc (Table 8). This indicates that BAFF blockade withBAFFR-Fc inhibits autoantibody production, which may contribute todecreased pathology in BAFFR-Fc-treated mice. Notably, autoantibodylevels remained decreased compared with controls 10 weeks (age 37 weeks)after the last dose of BAFFR-Fc had been administered.

TABLE 8 Anti-ssDNA (μg/ml) Age Treatment (weeks) BAFFR-Fc (n = 10) mlgG(n = 10) 23 0.1¹ 0.15 29 0.4 1.1 37 0.7 24.3 ¹Values reflect geometricmeans.

Example 6 Short-Term BAFFR-Fc Administration Reduces Percentage ofIgM⁻IgD⁺ B Cells

At the end of the study, BAFFR-Fc-treated and mIgG-treated mice had asimilar frequency of total B cells in the blood, based on staining forB220⁺ B cells. However, when specific B cell subsets were examined,BAFFR-Fc-treated mice exhibited a statistically significant reduction(p<0.01, Student's t test) in the percent of IgM⁻IgD⁺ B cells (Table 9).The general reconstitution of peripheral B cells, yet the selective,marked reduction in IgM⁻IgD⁺ B cells and long-term efficacy inBAFFR-Fc-treated mice suggests that BAFFR-Fc causes a long-termreduction in these presumably, pathogenic B cells.

TABLE 9 Percent blood B cells (mean ± SD) BAFFR-Fc¹ mlgG¹ IgM⁻IgD⁺ Bcells  8.7 ± 5.0 44.9 ± 7.3 Total B220⁺ B cells 47.3 ± 9.3 49.6 ± 8.6¹Mice were 49-51 weeks of age at the time of analysis.

The specification is most thoroughly understood in light of theteachings of the references cited within the specification. Theembodiments within the specification provide an illustration ofembodiments of the invention and should not be construed to limit thescope of the invention. The skilled artisan readily recognizes that manyother embodiments are encompassed by the invention. All publications andpatents and sequences cited in this disclosure are incorporated byreference in their entirety. To the extent the material incorporated byreference contradicts or is inconsistent with the present specification,the present specification will supercede any such material. The citationof any references herein is not an admission that such references areprior art to the present invention.

The following applies unless otherwise indicated: all numbers expressingquantities of ingredients, cell culture, treatment conditions, and soforth used in the specification, including claims, are to be understoodas being modified in all instances by the term “about”; the term “atleast” preceding a series of elements is to be understood to refer toevery element in the series. Those skilled in the art will recognize, orbe able to ascertain using no more than routine experimentation, manyequivalents to the specific embodiments of the invention describedherein. Such equivalents are intended to be encompassed by the followingclaims.

1. A method of treating a patient having an immunologic disorder, comprising: (a) administering to the patient a therapeutically effective amount of a BAFF (B cell activating factor belonging to the TNF family) antagonist at least once or at one or more intervals of less than N weeks; (b) temporarily discontinuing the administration of step (a) for N weeks or longer; and (c) repeating steps (a) and (b) at least once; wherein N is 8, 9, 10, 11, or
 12. 2. The method of claim 1, wherein the administration of step (a) comprises an interval of 1, 2, 3, 4, 5, 6, or 7 weeks.
 3. The method of claim 1, wherein the BAFF antagonist is administered in step (a) 2, 3, 4, 5, 6, or 7 times a week.
 4. The method of claim 1, wherein the administration is discontinued in step (b) for 12, 18, 24, 30, 36, 42, 48 weeks or longer.
 5. The method of claim 1, wherein at the beginning of the treatment the patient has one or more of: (i) proteinuria of 1 g per a 24-hour period or higher; (ii) serum creatinine levels of about 1 mg/dl or higher; (iii) creatinine clearance levels of 97 ml/min or lower; (iv) blood urea of 20 mg/dl or higher; (v) abnormal titer of autoantibodies in the serum; and (vi) peripheral blood B cell count of 700 cells/μl.
 6. The method of claim 5, wherein the patient is human.
 7. The method of claim 1, wherein the therapeutically effective amount of the BAFF antagonist is sufficient to inhibit autoantibody titer.
 8. The method of claim 1, wherein the therapeutically effective amount of the BAFF antagonist is sufficient to reduce B cell hyperplasia.
 9. The method of claim 1, wherein the therapeutically effective amount of the BAFF antagonist is sufficient to reduce cardiac inflammation.
 10. The method of claim 1, wherein the therapeutically effective amount of the BAFF antagonist is sufficient to improve renal function.
 11. The method of claim 10, wherein the renal function is one or more of: pressure filtration, selective reabsorption, tubular secretion, and systemic blood pressure regulation.
 12. The method of claim 1, wherein the therapeutically effective amount of the BAFF antagonist is sufficient to reduce progression of renal fibrosis.
 13. The method of claim 1, wherein the therapeutically effective amount of the BAFF antagonist is sufficient to reduce lymphocyte infiltration in the kidneys.
 14. The method of claim 1, wherein the therapeutically effective amount of the BAFF antagonist is sufficient to reduce lymphadenopathy.
 15. The method of claim 1, wherein the immunologic disorder is an autoimmune disorder.
 16. The method of claim 15, wherein the autoimmune disorder is systemic lupus erythematosus.
 17. The method of any one of claims 1-16, wherein the medicament is LymphoStat-B™.
 18. The method of any one of claims 1-16, wherein the BAFF antagonist is BAFF-specific.
 19. The method of claim 18, wherein the BAFF antagonist is selected from the group consisting of a soluble BAFF receptor, an anti-BAFF antibody and an anti-BAFFR antibody.
 20. The method of claim 19, wherein the BAFF receptor is selected from the group consisting of BAFFR, BCMA (B cell maturation antigen) and TACI (transmembrane activator and cyclophilin ligand interactor).
 21. The method of claim 20, wherein the soluble BAFFR comprises the peptide of SEQ ID NO:5.
 22. The method of claim 19, wherein the soluble BAFF receptor comprises a BAFF-binding domain of BAFFR.
 23. The method of claim 20, wherein the soluble BAFFR is human.
 24. The method of claim 20, wherein the soluble BAFFR lacks the sequence of SEQ ID NO:6.
 25. The method of claim 22, wherein the BAFF-binding domain of BAFFR has an amino acid sequence as set out: (a) from amino acid 27 to amino acid 32 of SEQ ID NO:1; (b) from amino acid 18 to amino acid 43 of SEQ ID NO:1; (c) from amino acid 13 to amino acid 50 of SEQ ID NO:1; (d) from amino acid 3 to amino acid 73 of SEQ ID NO:1; or (e) amino acid 2 to amino acid 62 of SEQ ID NO:3.
 26. The method of claim 22, wherein the BAFF-binding domain of BAFFR is fused to a constant region of an immunoglobulin.
 27. The method of claim 26, wherein the immunoglobulin is IgG₁ or IgG₄.
 28. The method of claim 26, wherein the constant region of an immunoglobulin comprises an Fc portion.
 29. The method of claim 28, wherein the BAFFR-Fc comprises (a) an amino acid sequence as set out in SEQ ID NO:2 or (b) an amino acid sequence as set out in SEQ ID NO:4.
 30. A method of treating a patient having an autoimmune disorder, comprising: (a) administering to the patient a therapeutically effective amount of a BAFF-specific antagonist at least once or at one or more intervals of less than N weeks; (b) temporarily discontinuing the administration of step (a) for N weeks or longer; and (c) repeating steps (a) and (b) at least once; thereby treating the autoimmune disorder, and wherein N is 8, 9, 10, 11, or
 12. 31. A method of reducing autoantibody titer in a patient, comprising: (a) administering to the patient a therapeutically effective amount of a BAFF-specific antagonist at least once or at one or more intervals of less than N weeks; (b) temporarily discontinuing the administration of step (a) for N weeks or longer; and (c) repeating steps (a) and (b) at least once; thereby reducing autoantibody titer, and wherein N is 8, 9, 10, 11, or
 12. 32. A method of inhibiting generation of pathogenic B cells in a patient, comprising: (a) administering to the patient a therapeutically effective amount of a BAFF-specific antagonist at least once or at one or more intervals of less than N weeks; (b) temporarily discontinuing the administration of step (a) for N weeks or longer; and (c) repeating steps (a) and (b) at least once; thereby inhibiting generation of pathogenic B cells, and wherein N is 8, 9, 10, 11, or
 12. 33. The method of claim 32, wherein the pathogenic B cells are IgM⁻IgD⁺.
 34. The method of any one of claims 30-33, wherein the BAFF-specific antagonist is a soluble form of BAFFR.
 35. The method of any one of claims 30-33, wherein the BAFF-specific antagonist is an anti-BAFF-antibody.
 36. Use of a BAFF antagonist for preparation of a medicament for treatment of an immunologic disorder, wherein the treatment regimen comprises: (a) administering a therapeutically effective amount of a BAFF (B cell activating factor belonging to the TNF family) antagonist at least once or at one or more intervals of less than N weeks; (b) temporarily discontinuing the administration of step (a) for N weeks or longer; and (c) repeating steps (a) and (b) at least once; wherein N is 8, 9, 10, 11, or
 12. 37. The use of claim 36, wherein the administration of step (a) comprises an interval of 1, 2, 3, 4, 5, 6, or 7 weeks.
 38. The use of claim 36, wherein the BAFF antagonist is administered in step (a) 2, 3, 4, 5, 6, or 7 times a week.
 39. The use of claim 36, wherein the administration is discontinued in step (b) for 12, 18, 24, 30, 36, 42, 48 weeks or longer.
 40. The use of claim 36, wherein at the beginning of the treatment the patient has one or more of: (i) proteinuria of 1 g per a 24-hour period or higher; (ii) serum creatinine levels of about 1 mg/dl or higher; (iii) creatinine clearance levels of 97 ml/min or lower; (iv) blood urea of 20 mg/dl or higher; (v) abnormal titer of autoantibodies in the serum; and (vi) peripheral blood B cell count of 700 cells/μl.
 41. The use of claim 40, wherein the patient is human.
 42. The use of claim 36, wherein the therapeutically effective amount of the BAFF antagonist is sufficient to inhibit autoantibody titer.
 43. The use of claim 36, wherein the therapeutically effective amount of the BAFF antagonist is sufficient to reduce B cell hyperplasia.
 44. The use of claim 36, wherein the therapeutically effective amount of the BAFF antagonist is sufficient to reduce cardiac inflammation.
 45. The use of claim 36, wherein the therapeutically effective amount of the BAFF antagonist is sufficient to improve renal function.
 46. The use of claim 45, wherein the renal function is one or more of: pressure filtration, selective reabsorption, tubular secretion, and systemic blood pressure regulation.
 47. The use of claim 36, wherein the therapeutically effective amount of the BAFF antagonist is sufficient to reduce progression of renal fibrosis.
 48. The use of claim 36, wherein the therapeutically effective amount of the BAFF antagonist is sufficient to reduce lymphocyte infiltration in the kidneys.
 49. The use of claim 36, wherein the therapeutically effective amount of the BAFF antagonist is sufficient to reduce lymphadenopathy.
 50. The use of claim 36, wherein the immunologic disorder is an autoimmune disorder.
 51. The use of claim 50, wherein the autoimmune disorder is systemic lupus erythematosus.
 52. The use of any one of claims 36-51, wherein the medicament is LymphoStat-B™.
 53. The use of any one of claims 36-51, wherein the BAFF antagonist is BAFF-specific.
 54. The use of claim 53, wherein the BAFF antagonist is selected from the group consisting of a soluble BAFF receptor, an anti-BAFF antibody, and an anti-BAFFR antibody.
 55. The use of claim 54, wherein the soluble BAFF receptor is selected from the group consisting of BAFFR, BCMA (B cell maturation antigen) and TACI (transmembrane activator and cyclophilin ligand interactor).
 56. The use of claim 55, wherein the BAFFR comprises the peptide of SEQ ID NO:5.
 57. The use of claim 54, wherein the soluble BAFF receptor comprises a BAFF-binding domain of BAFFR.
 58. The use of claim 55, wherein the BAFFR is human.
 59. The use of claim 55, wherein the BAFFR lacks the sequence of SEQ ID NO:6.
 60. The use of claim 57, wherein the BAFF-binding domain of BAFFR has an amino acid sequence as set out: (a) from amino acid 27 to amino acid 32 of SEQ ID NO:1; (b) from amino acid 18 to amino acid 43 of SEQ ID NO:1; (c) from amino acid 13 to amino acid 50 of SEQ ID NO:1; (d) from amino acid 3 to amino acid 73 of SEQ ID NO:1; or (e) amino acid 2 to amino acid 62 of SEQ ID NO:3.
 61. The use of claim 57, wherein the BAFF-binding domain of BAFFR is fused to a constant region of an immunoglobulin.
 62. The use of claim 61, wherein the immunoglobulin is IgG₁ or IgG₄.
 63. The use of claim 61, wherein the constant region of an immunoglobulin comprises an Fc portion.
 64. The use of claim 63, wherein the BAFFR-Fc comprises (a) an amino acid sequence as set out in SEQ ID NO:2 or (b) an amino acid sequence as set out in SEQ ID NO:4.
 65. Use of a BAFF-specific antagonist for preparation of a medicament for treatment of an autoimmune disorder, wherein the treatment regimen comprises: (a) administering to the patient a therapeutically effective amount of a BAFF-specific antagonist at least once or at one or more intervals of less than N weeks; (b) temporarily discontinuing the administration of step (a) for N weeks or longer; and (c) repeating steps (a) and (b) at least once; thereby treating an autoimmune disorder, and wherein. N is 8, 9, 10, 11, or
 12. 66. Use of a BAFF-specific antagonist for preparation of a medicament for reducing autoantibody titer, wherein the treatment regimen comprises: (a) administering to the patient a therapeutically effective amount of a BAFF-specific antagonist at least once or at one or more intervals of less than N weeks; (b) temporarily discontinuing the administration of step (a) for N weeks or longer; and (c) repeating steps (a) and (b) at least once; thereby reducing autoantibody titer, and wherein N is 8, 9, 10, 11, or
 12. 67. Use of a BAFF-specific antagonist for preparation of a medicament for inhibiting generation of pathogenic B cells, wherein the treatment comprises: (a) administering to a patient a therapeutically effective amount of a BAFF-specific antagonist at least once or at one or more intervals of less than N weeks; (b) temporarily discontinuing the administration of step (a) for N weeks or longer; and (c) repeating steps (a) and (b) at least once; wherein N is 8, 9, 10, 11, or
 12. 68. The use of claim 67, wherein the pathogenic B cells are IgM⁻IgD⁺.
 69. The use of any one of claims 65-68, wherein the BAFF-specific antagonist is a soluble form of BAFFR.
 70. The use of any one of claims 65-68, wherein the BAFF-specific antagonist is an anti-BAFF-antibody. 